Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

Germline specification underlies human reproduction and evolution, but it has proven difficult to study in humans since it occurs shortly after blastocyst implantation. This process can be modeled with human induced pluripotent stem cells (hiPSCs) by differentiating them into primordial germ cell-like cells (hPGCLCs) through an incipient mesoderm-like cell (iMeLC) state. Here, we elucidate the key transcription factors and their interactions with important signaling pathways in driving hPGCLC differentiation from iPSCs. Germline competence of iMeLCs is dictated by the duration and dosage of WNT signaling, which induces expression of EOMES to activate SOX17, a key driver of hPGCLC specification. Upon hPGCLC induction, BMP signaling activates TFAP2C in a SOX17-independent manner. SOX17 and TFAP2C then cooperatively instate an hPGCLC transcriptional program, including BLIMP1 expression. This specification program diverges from its mouse counterpart regarding key transcription factors and their hierarchies, and it provides a foundation for further study of human germ cell development.

Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.